Fluorometric analysis has been performed for the detection of primary and secondary amino acids from a single column. One such system is described in a paper by Felix and Terkelsen, entitled "Total Fluorometric Amino Acid Analysis using Fluorescamine," Archives of Biochemistry and Biophysics, Vol. 157, No. 1, July 1973. The same system is described in a paper by the same authors entitled "Determination of Hydroxyproline in Fluorometric Amino Acid Analysis with Fluorescamine," Analytical Biochemistry, Vol. 56, No. 2, December 1973, p. 610. In the above Felix et al articles, standard amino acid mixtures are eluted from a chromotographic column by the use of standard buffer solutions. Fluorescamine is added to the solution prior to passage through the fluorometer. When the sample stream contains secondary amino acid, an oxidizing agent, N-chlorosuccinimide (herein NCS) is added to the sample stream prior to reaction with the fluorescamine. This converts the secondary amino acid to a form which is detectable by the fluorometer upon reaction with the fluorescamine.
The secondary amino acids, e.g. proline and 4-hydroxyproline, of the sequentially eluted sample stream must be converted to another form to be rendered detectable by reaction with conventional fluorogens, e. g., fluorescamine or O-phthalaldehyde. One conversion technique is reaction with an oxidizing agent, N-chlorosuccinimide, as illustrated in the above Felix et al articles. However, the oxidizing agent is known to adversely affect the quantitative detection of the primary amino acids in the stream. Thus, the oxidizing agent conventionally is added to the reagent stream only when the secondary amino acid is present by "pulsing" the oxidizing agent into the stream at that time.
A disadvantage of the above system is that the pulsing causes a sudden increase in the flow rate of liquid through the fluorometer. In turn, this causes a marked "baseline" shift in the recorded chromatograms as illustrated in the foregoing papers. Complex electronics is required to compensate for this baseline shift which is the cause of potential analytical error, as by failure to properly compensate for the pulse in the calibration. The same problem is inherent in other fluorometric detection systems in which an oxidizing agent is pulsed during the presence of only a secondary amino acid.